MAMMALIAN S-ADO-MET DECARBOXYLASE
Byrd, R. A,, Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1977) J . Am. Chem. SOC.99,6139-6141. Byrd, R. A., Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1978) J . A m . Chem. SOC.100, 7478-7486. Cipollo, K. L., & Dunlap, R. B. (1978) Biochem. Biophys. Res. Commun. 81, 1139-1144. Daron, H. H., & Aull, J. L (1978) J . Biol. Chem. 253, 940-94 5. Donato, H., Aull, J. L., Lyon, J. A., Reinsch. J. W., & Dunlap, R. B. (1976) J . Biol. Chem. 251, 1303-1310. Dunlap, R. B., Harding, N. G. L., & Huennekens, F. M. (1971) Biochemistry I O , 88-97. Galivan, J. H., Maley, G. F., & Maley, F. (1975) Biochemistry 14, 3338-3344. Galivan, J., Maley, G. F., & Maley F. (1976a) Biochemistry 15, 356-362. Galivan, J. H., Maley, F., & Baugh, C. M. (1976b) Biochem. Biophys. Res. Commun. 71, 527-534. Galivan, J., Noonan, J., & Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345. Hatefi, Y., Talbert, P. J., Osborn, M. J., & Huennekens, F. M. (1960) Biochem. Prep. I , 89-92. Kisliuk, F. L., Gaumont, Y . , & Baugh, C. M. (1974) J . Biol. Chem. 249, 4100-4103. Lange, L. G., 111, Riordan, J. F., & Vallee, B. L. (1974) Biochemistry 13, 4361-4370.
VOL. 18, NO. 25, 1979
5541
Lewis, C. A,, Jr., Munroe, W. A., & Dunlap, R. B. (1978) Biochemistry 17, 5382-5387. Lyon, J. A., Pollard, A. L., Loeble, R. B., & Dunlap, R. B. (1975) Cancer Biochem. Biophys. I , 121-128. Matthews, D. A., Alden, R. A,, Bolin, J. T., Freer, S . T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M., & Hoogsteen, K. (1977) Science 197, 452-455. Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S . T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J . Biol. Chem. 253, 6946-6954. Plese, P. C., & Dunlap, R. B. (1977) J . Biol. Chem. 252, 6 139-6 144. Pogolotti, A. L., Ivanetich, K. M., Sommer, H., & Santi, D. V. (1976) Biochem. Biophys. Res. Commun. 70,972-978. Reyes, P., & Heidelberger (1965) Mol. Pharrnacol. I , 14-30. Riley, H. A., & Gray, A. R. (1943) Org. Synth. 2, 509-5 1 1 . Riordan, J. F. (1973) Biochemistry 12, 3915-3922. Riordan, J . F., McElvany, K. D., & Borders, C. L., Jr. (1977) Science 195, 884-886. Schubert, M. P. (1935) J . Biol. Chem. I l l , 671-678. Smith, D. J., Maggio, E. T., & Kenyon, G. L. (1975) Biochemistry 14, 766-771. Takahashi, K. (1968) J . Biol. Chem. 243, 6171-6179. Takahashi, K. (1977) J . Biochem. (Tokyo) 81, 395-402.
Purification and Characterization of S-Adenosyl-L-methionine Decarboxylase from Mouse Mammary Gland and Liver? Tadashi Sakai, Chiyo Hori, Kazutaka Kano, and Takami Oka*
ABSTRACT: S-Adenosyl-L-methionine decarboxylase, a key enzyme in polyamine biosynthesis, has been purified to apparent homogeneity from mouse mammary gland and liver by a combination of ammonium sulfate fractionation, DEAE-cellulose and methylglyoxal bis(guany1hydrazone)Sepharose 4B affinity chromatographies, and gel filtration. Crucial factors for good yield of the pure enzyme include the use of putrescine, pyridoxal phosphate, a detergent (0.01% deoxycholate), and high salts as stabilizing agents at various stages of purification. The purified enzyme from both tissues has essentially identical specific activities and other properties examined and is completely free of propylamine transferase activity (spermidine or spermine synthase). The apparent molecular weight of native enzyme as determined by gel filtration and by sucrose density centrifugation was approximately 68 000 and 74 000, respectively, whereas the subunit molecular weight of 32 000 was obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a dimer. The enzyme has an isoelectric point of 5.9 and a pH optimum of 7.5. The activity of purified enzyme is stimulated by physiological concentrations of putrescine ( K ,
= 0.5 pM), which decreases the apparent K , for the substrate from 1 . 1 X lo4 to 2 X M and also prevents inactivation of the enzyme. Another related polyamine, spermine, but not spermidine, inhibits the enzyme activity by reducing the V,, of the enzyme reaction at physiological concentrations (Ki= 0.5 mM). By use of the purified liver S-adenosyl-L-methionine decarboxylase, a monospecific mouse antibody to this enzyme has been raised in rabbits. The antibody cross-reacts with the enzyme from mammary gland, inactivates the enzyme, and forms a single precipitation line with S-adenosine-L-methionine decarboxylase from both tissues as shown by an Ouchterlony double-diffusion test. Immunoproduct analysis by sodium dodecyl sulfate gel electrophoresis of the antigen synthesized by mouse mammary explants in vitro demonstrated the presence of a sharp band which comigrated with the subunit of authentic (pure) enzyme. The antibody was used in measuring levels of antigen in the mammary gland of mice treated with methylglyoxal bis(guanylhydrazone), and the results indicated that the increased accumulation of the enzyme activity was primarily due to alterations in synthesis and degradation of enzyme molecules.
S-Adenosyl-L-methionine decarboxylase (S-Ado-Met decarboxylase) l serves an essential role for polyamine biosynthesis by catalyzing the formation of S-methyladenosy1-L-
homocysteamine (decarboxylated S-Ado-Met), the sole donor of the propylamine moiety for the biosynthesis of spermidine
t From the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205. Receiued June 7, 1979.
S-Ado-Met, S-adenosyl-L-methionine; NaDodSO,, sodium dodecyl sulfate; BSA, bovine serum albumin; ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase.
’ Abbreviationsused: MGBG, methyl glyoxal bis(guany1hydrazone); ~~
This article not subject to US.Copyright. Published 1979 by the American Chemical Society
5542
BIOCHEMISTRY
and spermine (Pegg & Williams-Ashman, 1969; WilliamsAshman et al., 1972; Tabor & Tabor, 1972). Earlier studies on S-Ado-Met decarboxylase in mammary gland have shown that the enzyme activity increases in parallel with the rise in the intracellular concentration of spermidine during the development of mammary epithelium that occurs in lactating rats (Russell & McVicker, 1972) and mice (Oka et al., 1978) as well as during the hormonally induced development of mouse mammary gland in culture (Oka & Perry, 1974; Sakai et al., 1978). Further studies on the organ culture system with an inhibitor of S-Ado-Met decarboxylase, methylglyoxal bis(guany1hydrazone) (MGBG), have indicated (Oka & Perry, 1974; Rillema, 1976; Sakai et al., 1978) that S-Ado-Met decarboxylase may be a rate-limiting enzyme for the biosynthesis of spermidine, which plays a key role for the hormone-dependent development of mouse mammary epithelium. For facilitation of our previous attempts to elucidate the cellular mechanism involved in the regulation of S-Ado-Met decarboxylase in the development of mouse mammary epithelium, a specific antibody probe of the enzyme would be a valuable tool for direct quantitative analysis. Thus, we have undertaken the purification of mouse S-Ado-Met decarboxylase and report here the isolation of the homogeneous enzyme, its characterization, and the preparation of antibody against the protein. Materials and Methods
Materials Materials were purchased as follows: [carboxy-I4C]-Sadenosyl-L-methionine (54.6 mCi/mmol), [ 1,4-14C]putrescine dihydrochloride (51.8 mCi/mmol), [ 14C]spermidine trihydrochloride (10.0 mCi/mmol), and [3,4,5-'H3]1eucine from New England Nuclear; S-adenosyl-L-methionine, dithiothreitol, ammonium sulfate, and sucrose from Schwartz/ Mann; putrescine dihydrochloride, pyridoxal phosphate, cadaverine, and L-ornithine from Calbiochem; spermidine, spermine, N-ethylmaleimide, and p-(ch1oromercuri)benzoate from Sigma Chemical Co.; MGBG from Aldrich Chemical Co.; DEAE-cellulose (DE-52) from Whatman, Inc. Marker proteins for molecular weight determination, such as aldolase, bovine serum albumin, ovalbumin, chymotrypsinogen, cytochrome c, and ferritin, were purchased from Boehringer Mannheim Corp. and Pharmacia Fine Chemicals, Inc. CHSepharose 4B and Sephacryl S-200 were obtained from Pharmacia Fine Chemicals, Inc. Medium 199 and phosphate-buffered saline were obtained from GIBCO. Female C3H/HeN mice in the fifth to tenth day of lactation were obtained from the National Institutes of Health. Decarboxylated S-adenosyl-L-methionine was synthesized enzymatically by the method of Tabor (1962) with partially purified adenosylmethionine decarboxylase from Escherichia coli. The product was isolated from the reaction mixture by a Dowex 50 column and purified to homogeneity by preparative paper electrophoresis. Preparation of CH-Sepharose 4B containing covalently bound MGBG was carried out by using carbodiimide (Sigma) according to the method of Pegg ( 1 974). Methods Purification of S-Ado-Met Decarboxylase from Mouse Mammary Gland and Liver. All mice used were given an intraperitoneal injection of 2 mg of MGBG in 0.2 mL of 0.1 5 M NaC1. This treatment was given to increase the level of the enzyme in mouse liver and mammary gland by 10-20-fold, as reported in other species (Pegg, 1974; Heby et al., 1973; Poso et al., 1975). Twenty-four hours after the injection, the
SAKAI, H O R I , KANO, A N D OKA
animals were killed by cervical dislocation, and the liver and mammary gland were removed. The tissues were minced, washed in 0.15 M NaC1, and homogenized with a PotterElvehjem homogenizer in 4 volumes of ice-cold 25 mM sodium phosphate buffer, pH 7.6, which contained 1 mM dithiothreitol, 0.1 mM EDTA, 0.5 mM putrescine, and 5 1 M pyridoxal phosphate (buffer A), unless stated otherwise. All operations were carried out at 0-4 "C. The homogenate was centrifuged at 24000g for 60 min, and the supernatant was saved. The precipitate was rehomogenized with 10-15 mL of buffer A and centrifuged again in the same manner. A portion of the combined supernatant fluids was removed, dialyzed against buffer A to remove MGBG, and assayed for enzyme activity. The remaining supernatant fluid was fractionated by stepwise addition of solid ammonium sulfate. Proteins precipitating between 35 and 65% saturation were collected by centrifugation at 25000g for 1 h and then dissolved in 10-15 mL of buffer A and dialyzed against 100 volumes of the same buffer for 12 h. The dialyzed enzyme solution was applied to a DEAE-52 cellulose column (2.5 X 20 cm) which had been previously equilibrated with buffer A. After the column was washed with 200 mL of buffer A, S-Ado-Met decarboxylase was eluted with a linear gradient of 0-0.4 M NaCl in buffer A. The total volume of the gradient solution was 800 mL. All fractions containing S-Ado-Met decarboxylase were pooled and reprecipitated with 65% ammonium sulfate. The precipitate was dissolved in 10 mM Tris-HC1 buffer, pH 7.6 (buffer B), containing all the substances present in buffer A except sodium phosphate and dialyzed against 1000 volumes of the same buffer. This change of the buffer system was necessary since the phosphate buffer formed insoluble materials with MGBG which was used in the subsequent affinity chromatography step. The dialyzed enzyme solution was applied to a column (1 X 10 cm) of Sepharose 4B to which MGBG was covalently bound and which had been previously equilibrated with buffer B. The column was washed with 100 mL of buffer B and then with 100 mL of 0.3 M NaCl in the same buffer, and S-Ado-Met decarboxylase activity was eluted from the column with buffer B containing 0.3 M NaCl and 1 mM MGBG. During the elution process, each fraction was collected in a tube containing one-tenth volume of 0.1% deoxycholate, which was found to be necessary to prevent the loss of the enzyme activity. In the absence of the detergent, recovery of the enzyme activity was reduced to less than 1oOh of the activity applied to the column. Each 1-mL fraction was dialyzed against 1000 volumes of buffer B containing 0.0 1% deoxycholate with three buffer changes over a 24-h period. Fractions containing S-Ado-Met decarboxylase activity were pooled and concentrated by an Amicon Centriflo membrane cone (Amicon F25) to -0.5 mL. The enzyme solution was applied to a column (0.7 X 55 cm) of Sephacryl S-200 which had been previously equilibrated with buffer B containing 0.5 M NaCl and 0.01% deoxycholate. Following elution with the same buffer, each fraction was assayed for the enzyme activity and the active fractions were pooled and stored at 4 OC without appreciable loss of activity within a 1-month period. Molecular Weight Determinations. The molecular weight of the native enzyme was determined under nondenaturing conditions by sucrose density centrifugation (Martin & Ames, 1961) and by Sephacryl S-200 gel filtration. An analytical column of Sephacryl S-200 (1.1 X 44 cm) was calibrated with proteins of known molecular weights. Analytical Methods. The activity of S-Ado-Met decarboxylase was assayed by measuring I4CO2release from
MAMMALIAN S-ADO-MET DECARBOXYLASE
VOL. 1 8 , N O . 2 5 , 1919
5543
Table I: Summary of Purification Procedure for S-AdeMet Decarboxylase from Mouse Mammary Gland and Livera
S-AdeMet decarboxylase step
total protein (mg)
total act. (nmol/h)
sp act. recovery [nmol/(mg h)] (%)
purifn (x-fold)
spermidine synthase, sp act. [pmol/(mg h)]
( 1 ) 25000g supernatant (2) ammonium sulfate ppt (3565%) (3) DEAE column chromatography (4) MGBG affinity column chromatography (5) Sephacryl gel filtration
Mammary Gland 1653 1607 524 1389 168 1089 0.0514 251 0.0327 220
0.972 2.65 6.48 4892 6724
100 86 68 16 14
1 2.73 6.67 5033 6918
240 820 1490 0
(1) 25000g supernatant (2) ammonium sulfate ppt (35-65%) (3) DEAE column chromatography (4) MGBG affinity column chromatography (5) Sephacryl gel filtration
Liver 19266 1720 5800 1233 342 1284 0.0540 267 0.0369 226
0.0893 0.213 3156 4944 6120
100 72 75 16 13
1 2.39 42.1 55363 68533
290 960 2410
Byrd, R. A,, Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1977) J . Am. Chem. SOC.99,6139-6141. Byrd, R. A., Dawson, W. H., Ellis, P. D., & Dunlap, R. B. (1978) J . A m . Chem. SOC.100, 7478-7486. Cipollo, K. L., & Dunlap, R. B. (1978) Biochem. Biophys. Res. Commun. 81, 1139-1144. Daron, H. H., & Aull, J. L (1978) J . Biol. Chem. 253, 940-94 5. Donato, H., Aull, J. L., Lyon, J. A., Reinsch. J. W., & Dunlap, R. B. (1976) J . Biol. Chem. 251, 1303-1310. Dunlap, R. B., Harding, N. G. L., & Huennekens, F. M. (1971) Biochemistry I O , 88-97. Galivan, J. H., Maley, G. F., & Maley, F. (1975) Biochemistry 14, 3338-3344. Galivan, J., Maley, G. F., & Maley F. (1976a) Biochemistry 15, 356-362. Galivan, J. H., Maley, F., & Baugh, C. M. (1976b) Biochem. Biophys. Res. Commun. 71, 527-534. Galivan, J., Noonan, J., & Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345. Hatefi, Y., Talbert, P. J., Osborn, M. J., & Huennekens, F. M. (1960) Biochem. Prep. I , 89-92. Kisliuk, F. L., Gaumont, Y . , & Baugh, C. M. (1974) J . Biol. Chem. 249, 4100-4103. Lange, L. G., 111, Riordan, J. F., & Vallee, B. L. (1974) Biochemistry 13, 4361-4370.
VOL. 18, NO. 25, 1979
5541
Lewis, C. A,, Jr., Munroe, W. A., & Dunlap, R. B. (1978) Biochemistry 17, 5382-5387. Lyon, J. A., Pollard, A. L., Loeble, R. B., & Dunlap, R. B. (1975) Cancer Biochem. Biophys. I , 121-128. Matthews, D. A., Alden, R. A,, Bolin, J. T., Freer, S . T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M., & Hoogsteen, K. (1977) Science 197, 452-455. Matthews, D. A., Alden, R. A., Bolin, J. T., Filman, D. J., Freer, S . T., Hamlin, R., Hol, W. G. J., Kisliuk, R. L., Pastore, E. J., Plante, L. T., Xuong, N., & Kraut, J. (1978) J . Biol. Chem. 253, 6946-6954. Plese, P. C., & Dunlap, R. B. (1977) J . Biol. Chem. 252, 6 139-6 144. Pogolotti, A. L., Ivanetich, K. M., Sommer, H., & Santi, D. V. (1976) Biochem. Biophys. Res. Commun. 70,972-978. Reyes, P., & Heidelberger (1965) Mol. Pharrnacol. I , 14-30. Riley, H. A., & Gray, A. R. (1943) Org. Synth. 2, 509-5 1 1 . Riordan, J. F. (1973) Biochemistry 12, 3915-3922. Riordan, J . F., McElvany, K. D., & Borders, C. L., Jr. (1977) Science 195, 884-886. Schubert, M. P. (1935) J . Biol. Chem. I l l , 671-678. Smith, D. J., Maggio, E. T., & Kenyon, G. L. (1975) Biochemistry 14, 766-771. Takahashi, K. (1968) J . Biol. Chem. 243, 6171-6179. Takahashi, K. (1977) J . Biochem. (Tokyo) 81, 395-402.
Purification and Characterization of S-Adenosyl-L-methionine Decarboxylase from Mouse Mammary Gland and Liver? Tadashi Sakai, Chiyo Hori, Kazutaka Kano, and Takami Oka*
ABSTRACT: S-Adenosyl-L-methionine decarboxylase, a key enzyme in polyamine biosynthesis, has been purified to apparent homogeneity from mouse mammary gland and liver by a combination of ammonium sulfate fractionation, DEAE-cellulose and methylglyoxal bis(guany1hydrazone)Sepharose 4B affinity chromatographies, and gel filtration. Crucial factors for good yield of the pure enzyme include the use of putrescine, pyridoxal phosphate, a detergent (0.01% deoxycholate), and high salts as stabilizing agents at various stages of purification. The purified enzyme from both tissues has essentially identical specific activities and other properties examined and is completely free of propylamine transferase activity (spermidine or spermine synthase). The apparent molecular weight of native enzyme as determined by gel filtration and by sucrose density centrifugation was approximately 68 000 and 74 000, respectively, whereas the subunit molecular weight of 32 000 was obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a dimer. The enzyme has an isoelectric point of 5.9 and a pH optimum of 7.5. The activity of purified enzyme is stimulated by physiological concentrations of putrescine ( K ,
= 0.5 pM), which decreases the apparent K , for the substrate from 1 . 1 X lo4 to 2 X M and also prevents inactivation of the enzyme. Another related polyamine, spermine, but not spermidine, inhibits the enzyme activity by reducing the V,, of the enzyme reaction at physiological concentrations (Ki= 0.5 mM). By use of the purified liver S-adenosyl-L-methionine decarboxylase, a monospecific mouse antibody to this enzyme has been raised in rabbits. The antibody cross-reacts with the enzyme from mammary gland, inactivates the enzyme, and forms a single precipitation line with S-adenosine-L-methionine decarboxylase from both tissues as shown by an Ouchterlony double-diffusion test. Immunoproduct analysis by sodium dodecyl sulfate gel electrophoresis of the antigen synthesized by mouse mammary explants in vitro demonstrated the presence of a sharp band which comigrated with the subunit of authentic (pure) enzyme. The antibody was used in measuring levels of antigen in the mammary gland of mice treated with methylglyoxal bis(guanylhydrazone), and the results indicated that the increased accumulation of the enzyme activity was primarily due to alterations in synthesis and degradation of enzyme molecules.
S-Adenosyl-L-methionine decarboxylase (S-Ado-Met decarboxylase) l serves an essential role for polyamine biosynthesis by catalyzing the formation of S-methyladenosy1-L-
homocysteamine (decarboxylated S-Ado-Met), the sole donor of the propylamine moiety for the biosynthesis of spermidine
t From the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205. Receiued June 7, 1979.
S-Ado-Met, S-adenosyl-L-methionine; NaDodSO,, sodium dodecyl sulfate; BSA, bovine serum albumin; ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase.
’ Abbreviationsused: MGBG, methyl glyoxal bis(guany1hydrazone); ~~
This article not subject to US.Copyright. Published 1979 by the American Chemical Society
5542
BIOCHEMISTRY
and spermine (Pegg & Williams-Ashman, 1969; WilliamsAshman et al., 1972; Tabor & Tabor, 1972). Earlier studies on S-Ado-Met decarboxylase in mammary gland have shown that the enzyme activity increases in parallel with the rise in the intracellular concentration of spermidine during the development of mammary epithelium that occurs in lactating rats (Russell & McVicker, 1972) and mice (Oka et al., 1978) as well as during the hormonally induced development of mouse mammary gland in culture (Oka & Perry, 1974; Sakai et al., 1978). Further studies on the organ culture system with an inhibitor of S-Ado-Met decarboxylase, methylglyoxal bis(guany1hydrazone) (MGBG), have indicated (Oka & Perry, 1974; Rillema, 1976; Sakai et al., 1978) that S-Ado-Met decarboxylase may be a rate-limiting enzyme for the biosynthesis of spermidine, which plays a key role for the hormone-dependent development of mouse mammary epithelium. For facilitation of our previous attempts to elucidate the cellular mechanism involved in the regulation of S-Ado-Met decarboxylase in the development of mouse mammary epithelium, a specific antibody probe of the enzyme would be a valuable tool for direct quantitative analysis. Thus, we have undertaken the purification of mouse S-Ado-Met decarboxylase and report here the isolation of the homogeneous enzyme, its characterization, and the preparation of antibody against the protein. Materials and Methods
Materials Materials were purchased as follows: [carboxy-I4C]-Sadenosyl-L-methionine (54.6 mCi/mmol), [ 1,4-14C]putrescine dihydrochloride (51.8 mCi/mmol), [ 14C]spermidine trihydrochloride (10.0 mCi/mmol), and [3,4,5-'H3]1eucine from New England Nuclear; S-adenosyl-L-methionine, dithiothreitol, ammonium sulfate, and sucrose from Schwartz/ Mann; putrescine dihydrochloride, pyridoxal phosphate, cadaverine, and L-ornithine from Calbiochem; spermidine, spermine, N-ethylmaleimide, and p-(ch1oromercuri)benzoate from Sigma Chemical Co.; MGBG from Aldrich Chemical Co.; DEAE-cellulose (DE-52) from Whatman, Inc. Marker proteins for molecular weight determination, such as aldolase, bovine serum albumin, ovalbumin, chymotrypsinogen, cytochrome c, and ferritin, were purchased from Boehringer Mannheim Corp. and Pharmacia Fine Chemicals, Inc. CHSepharose 4B and Sephacryl S-200 were obtained from Pharmacia Fine Chemicals, Inc. Medium 199 and phosphate-buffered saline were obtained from GIBCO. Female C3H/HeN mice in the fifth to tenth day of lactation were obtained from the National Institutes of Health. Decarboxylated S-adenosyl-L-methionine was synthesized enzymatically by the method of Tabor (1962) with partially purified adenosylmethionine decarboxylase from Escherichia coli. The product was isolated from the reaction mixture by a Dowex 50 column and purified to homogeneity by preparative paper electrophoresis. Preparation of CH-Sepharose 4B containing covalently bound MGBG was carried out by using carbodiimide (Sigma) according to the method of Pegg ( 1 974). Methods Purification of S-Ado-Met Decarboxylase from Mouse Mammary Gland and Liver. All mice used were given an intraperitoneal injection of 2 mg of MGBG in 0.2 mL of 0.1 5 M NaC1. This treatment was given to increase the level of the enzyme in mouse liver and mammary gland by 10-20-fold, as reported in other species (Pegg, 1974; Heby et al., 1973; Poso et al., 1975). Twenty-four hours after the injection, the
SAKAI, H O R I , KANO, A N D OKA
animals were killed by cervical dislocation, and the liver and mammary gland were removed. The tissues were minced, washed in 0.15 M NaC1, and homogenized with a PotterElvehjem homogenizer in 4 volumes of ice-cold 25 mM sodium phosphate buffer, pH 7.6, which contained 1 mM dithiothreitol, 0.1 mM EDTA, 0.5 mM putrescine, and 5 1 M pyridoxal phosphate (buffer A), unless stated otherwise. All operations were carried out at 0-4 "C. The homogenate was centrifuged at 24000g for 60 min, and the supernatant was saved. The precipitate was rehomogenized with 10-15 mL of buffer A and centrifuged again in the same manner. A portion of the combined supernatant fluids was removed, dialyzed against buffer A to remove MGBG, and assayed for enzyme activity. The remaining supernatant fluid was fractionated by stepwise addition of solid ammonium sulfate. Proteins precipitating between 35 and 65% saturation were collected by centrifugation at 25000g for 1 h and then dissolved in 10-15 mL of buffer A and dialyzed against 100 volumes of the same buffer for 12 h. The dialyzed enzyme solution was applied to a DEAE-52 cellulose column (2.5 X 20 cm) which had been previously equilibrated with buffer A. After the column was washed with 200 mL of buffer A, S-Ado-Met decarboxylase was eluted with a linear gradient of 0-0.4 M NaCl in buffer A. The total volume of the gradient solution was 800 mL. All fractions containing S-Ado-Met decarboxylase were pooled and reprecipitated with 65% ammonium sulfate. The precipitate was dissolved in 10 mM Tris-HC1 buffer, pH 7.6 (buffer B), containing all the substances present in buffer A except sodium phosphate and dialyzed against 1000 volumes of the same buffer. This change of the buffer system was necessary since the phosphate buffer formed insoluble materials with MGBG which was used in the subsequent affinity chromatography step. The dialyzed enzyme solution was applied to a column (1 X 10 cm) of Sepharose 4B to which MGBG was covalently bound and which had been previously equilibrated with buffer B. The column was washed with 100 mL of buffer B and then with 100 mL of 0.3 M NaCl in the same buffer, and S-Ado-Met decarboxylase activity was eluted from the column with buffer B containing 0.3 M NaCl and 1 mM MGBG. During the elution process, each fraction was collected in a tube containing one-tenth volume of 0.1% deoxycholate, which was found to be necessary to prevent the loss of the enzyme activity. In the absence of the detergent, recovery of the enzyme activity was reduced to less than 1oOh of the activity applied to the column. Each 1-mL fraction was dialyzed against 1000 volumes of buffer B containing 0.0 1% deoxycholate with three buffer changes over a 24-h period. Fractions containing S-Ado-Met decarboxylase activity were pooled and concentrated by an Amicon Centriflo membrane cone (Amicon F25) to -0.5 mL. The enzyme solution was applied to a column (0.7 X 55 cm) of Sephacryl S-200 which had been previously equilibrated with buffer B containing 0.5 M NaCl and 0.01% deoxycholate. Following elution with the same buffer, each fraction was assayed for the enzyme activity and the active fractions were pooled and stored at 4 OC without appreciable loss of activity within a 1-month period. Molecular Weight Determinations. The molecular weight of the native enzyme was determined under nondenaturing conditions by sucrose density centrifugation (Martin & Ames, 1961) and by Sephacryl S-200 gel filtration. An analytical column of Sephacryl S-200 (1.1 X 44 cm) was calibrated with proteins of known molecular weights. Analytical Methods. The activity of S-Ado-Met decarboxylase was assayed by measuring I4CO2release from
MAMMALIAN S-ADO-MET DECARBOXYLASE
VOL. 1 8 , N O . 2 5 , 1919
5543
Table I: Summary of Purification Procedure for S-AdeMet Decarboxylase from Mouse Mammary Gland and Livera
S-AdeMet decarboxylase step
total protein (mg)
total act. (nmol/h)
sp act. recovery [nmol/(mg h)] (%)
purifn (x-fold)
spermidine synthase, sp act. [pmol/(mg h)]
( 1 ) 25000g supernatant (2) ammonium sulfate ppt (3565%) (3) DEAE column chromatography (4) MGBG affinity column chromatography (5) Sephacryl gel filtration
Mammary Gland 1653 1607 524 1389 168 1089 0.0514 251 0.0327 220
0.972 2.65 6.48 4892 6724
100 86 68 16 14
1 2.73 6.67 5033 6918
240 820 1490 0
(1) 25000g supernatant (2) ammonium sulfate ppt (35-65%) (3) DEAE column chromatography (4) MGBG affinity column chromatography (5) Sephacryl gel filtration
Liver 19266 1720 5800 1233 342 1284 0.0540 267 0.0369 226
0.0893 0.213 3156 4944 6120
100 72 75 16 13
1 2.39 42.1 55363 68533
290 960 2410
